We have shown that the suppressor cells generated in an anti-non-H-2 mixed lymphocyte reaction (MLR) are Thy 1+, Lyt 1+2-, I-J-. The precursors of these cells in normal spleen express the same phenotype. We have preliminary evidence indicating that the suppressor cell also expresses Qa-1 alloantigens. It was shown, using a limiting dilution technique, that it was possible to dilute out such suppressor cells and thus observe a primary cytotoxic T-cell (Tc) response to non-H-2 alloantigens. In normal mice, such Tc responses to non-H-2 alloantigens have been demonstrated only previously using responder cells from immunized mice. We currently are investigating whether these primary responses are H-2 restricted as are the secondary responses and if the anti-non-H-2 Tc express the same Lyt phenotype as anti-non-h-2 Tc. We have also recently investigated the role of Ia antigens in the in vitro response of murine T-lymphpocytes to M1s antigens. We found that both proliferating and interleukin-2-producing T cells, which may or may not be the same cells at different stages of activation, require an Ia+ cell (and probably Ia antigens themselves) in the stimulator population in order to respond to M1s gene-encoded products. We are actively attempting to generate monoclonal antibodies to M1s-encoded molecules in order to investigate the molecular nature of these antigens and their possible topological relationship to Ia antigens on the cell surface. We have shown that antitumor (syngeneic to the responder strain) cytotoxic cells generated in an allogeneic (anti-H-2) MLR to be heterogeneous with respect to Lyt and Thy-1 phenotype. These cells express the following three phenotypes: (a)\Thy 1+, Lyt 2+; (b)\Thy 1+, Lyt 2-; and(c)\Thy 1-, Lyt 2-. We plan to further analyze the complexity of these MLR-activated tumor killer cells using antibodies to other differentiation antigens (Qa 5 for example) by both positive and negative selection procedures. It is also our aim to elucidate the pathways of their activation (i.e., the possible role of soluble mediators) and regulation by suppressor cells for which we have preliminary evidence.